Anti-inflammatory hydrolysate of C. versicolor

ABSTRACT

Methods and compositions for reducing and/or inhibiting inflammation by topical application of dermatocosmetic compositions comprising effective amounts of extracts of  Coriolus versicolor  that have been hydrolyzed by an acid protease, preferably  Rhizomucor miehei,  and thereafter rendered substantially devoid of acid-protease activity.

FIELD OF INVENTION

The present invention relates to methods and compositions for reducingand/or inhibiting topical inflammation. More particularly, the presentinvention relates to topical application of dermatocosmetic productscomprising effective amounts of extracts of Coriolus versicolor thathave been hydrolyzed by an acid protease, preferably Rhizomucor miehei,and thereafter rendered substantially devoid of acid-protease activity.

BACKGROUND OF THE INVENTION

Prostaglandins are potent mediators of a wide range of physiologicalprocesses, including inflammation. Cyclooxygenase (COX) enzymes, alsoreferred to as prostaglandin-endoperoxide synthases (PTGS), catalyze therate limiting step in prostaglandin synthesis. Of the two cyclooxygenaseenzymes, COX-2 enzyme (PTGS2) is inducible; it is produced in responseto specific homeostatic changes. The discovery that COX-2 regulates theproduction of prostaglandins involved in the inflammation process, leadto the development of a class of agents that function as COX-2inhibitors. Indeed, many anti-inflammatory agents now in the modernpharmacopoeia, including COX-2 inhibitors, have their origin in thepractices of traditional medicine where plants and their extracts wereadministered to provide relief from pain, fever and inflammation. Forexample, the discovery that the active analgesic ingredients in Willowbark are salicylates, lead to the syntheses of acetylsalicylic acid(aspirin) and numerous other non-steroidal anti-inflammatory drugs(NSAIDs). Oral administration of these compounds is widely known toproduce negative sequelae including irritation of the GI tract andbleeding. There has been, and remains a need, for efficacious topicalanti-inflammatory compounds. With increased awareness of the potency ofnaturally-derived active agents, the need for such compounds is evenmore strongly felt.

The present invention is directed to a novel proteinaceouspolysachharide extract of Coriolus versicolor, a mushroom with a historyboth in traditional Chinese botanical remedies as well as in modernbiomedicine as an adjunct in cancer treatments. As described in furtherdetail below, the compositions of the present invention are made by aheretofore unknown process in which proteinaceous polysaccharidesextracted from the fruiting body of C. versicolor are solubilized in anaqueous solvent and then hydrolyzed with a specific acid protease enzymederived from R. miehei, thus forming an extract with novelanti-inflammatory properties—specifically, the ability to reduce theexpression of mRNA coding for PTGS2 by at least about 67% as measured byDNA microarray analysis using a full-thickness epidermal skin tissuemodel.

Two peptidic polysaccharides derived from Coriolus versicolor have beenwidely-studied for their antiproliferative and antitumor activities. Thetwo protein-bound polysaccharides, designated as PSK and PSP, areisolated from the CM-101 and COV-1 strains of C. versicolor,respectively. PSP and PSK are chemically similar—both have molecularweights of approximately 100 kDa; both have glutamic acid and asparticacid as the two most abundant amino acid residues; and both havemonosaccharides with α-1,4 and β-1,3 glucosidic linkages. The twocompounds differ in that PSK contains fucose, while PSP containsrhamnose and arabinose. See, TB Ng. “A review of research on theprotein-bound polysaccharide (polysaccharo-peptide, PSP) from themushroom Coriolus versicolor (Basidiomycetes: Polyporaceae)” GenPharmacol Vol. 30, pp. 1-4 (1998).

PSK and PSP have also reported in the literature to haveimmuno-stimulatory effects. See TB Ng (1998) supra; see also Y. Dong, etal. “Antitumor effects of a refined polysaccharide peptide fractionisolated from Coriolus versicolor: in vitro and in vivo studies.” Res.Commun. Mol. Pathol. Pharmacol. Vol. 92, pp. 140-148 (1996). PSK iscommercially available under the tradename Krestin from Sankyo Co. Ltd.(Tokyo, Japan). See also, K K Chu, S S Ho and A H Chow, J. Clin.Pharmacol. Vol. 42, pp. 976-984 (2002)(describing traditional usage,pharmacological activities, clinical effects, adverse and reactions,active constituents of C. versicolor, J. Cui and Y. Chisti,Biotechnology Advances, Vol. 21, pp. 109-122 (review of thephysiological activity, uses and methods for producingpolysaccharopeptides from C. versiciolor).

While the prior art patent literature describes a number of processesfor preparing polysachharide polypeptide fractions from C. versicolor,these references neither teach nor suggest the methods for producing thecompounds of the present invention, nor the use of the product of thisnovel process (i.e., as a potent topical anti-inflammatory agent).

U.S. Pat. No. 4,051,314 is directed to oral administration ofdeproteinized polysaccharide hydrozylate derived from C. versicolor thatproduces anticarcinogenic activity in mice. Compositions claimed in thepresent invention are proteinaceous hydrolyzates.

U.S. Pat. No. 4,761,402 teaches use of PSK as a starting material forthe preparation of a lyophilized soluble phosphorylated glucan that canbe topically applied to treat infectious disease in animals or humans.The '402 Patent describes a soluble phosphorylated glucan prepared by aprocess comprising the steps of denaturing the three dimensionalstructure of a particulate glucan derived from C. versicolor followed byphosphorylation (mixing with phosphoric acid at a temperature of about100° C. for several hours).

U.S. Pat. No. 4,818,752 describes the use of soluble phosphorylatedglucan from C. versicolor prepared by the same process described in the'402 Patent and its use in treating malignant neoplastic disease inanimals and humans.

U.S. Pat. No. 4,833,131 describes a soluble phosphorylated glucan fromC. versicolor prepared by the same process described in the '402 Patentand its use in treating dermal wounds in animals and humans.

U.S. Pat. No. 5,374,714 describes a method of obtaining purifiedpolypeptides from the COV-1 strain of C. versicolor by a specificprocess—High Performance Liquid Chromatography (HPLC) using an acidsolvent including KCl in a reversed-phase column after extracting thematerials from the first and last chromatograph peaks. The '714 Patentdescribes purified polypeptides from this process as having cytotoxiceffects on human tumor cell lines as well as immunopotentiating effects,namely increasing white blood cells, T and B lymphocytes, and IgG. Thecompounds of the present invention are made by a process different thandescribed in the '714 Patent, including a different starting material.The '714 Patent does not suggest dermal application of the specifiedpolypeptides, nor use as topical agent that reduces the appearance ofchronological or environmentally caused aging.

U.S. Pat. Nos. 6,087,335 and 5,824,648 are related to the '714 Patentand claim polypetides derived from the COV-1 strain of C. versicolorhaving a specific partial amino acid sequence.

U.S. Pat. No. 4,202,969 describes low molecular weight (less than 5,000kDa) nitrogen-containing polysaccharide compounds having anti-tumoractivity extracted from C. versicolor by using a dilute alkalinesolvent.

U.S. Pat. No. 4,851,395 describes a nitrogen-containing polysaccharidesubstantially free of units having a molecular weight below about 5,000kDa produced by extracting C. versicolor with an aqueous alkalinesolution (0.01 to 2.0N) at a temperature of from 50° C. to 100° C.,neutralizing the resultant extract, followed by ultrafiltration orreverse osmosis to remove substantially all polymer units having amolecular weight below about 5,000 kDa.

U.S. Pat. Nos. 4,877,777, 4,900,722, and 4,975,421 assigned toBioGlucans, LP describe processes for preparing phosphorylated glucansfrom approximately two dozen source materials, including C. versicolor,in which the poly-[β-(1-3) glucopryanose]chains are phosphorylated invarying degrees by treating neutral glucans with phosphoric acid.

US Patent Application 2004/0137012 describes a pharmaceutically activeagent having a molecular weight of from about 5,000 to about 20,000useful in the treatment of diabetes where the agent is the product ofhydrolysis of β(1→3) glucan derived from vegetable material, includingCoriolus.

U.S. Pat. No. 4,614,733 discloses polysaccharides derived from C.versicolor having specific characteristics, including, molecular weight(from 5,000 to 300,000), infrared and nuclear magnetic resonanceabsorption spectra and solubility profile (soluble in water butinsoluble in pyridine, chloroform, and hexane). The polysaccharidestaught in the '733 Patent are made by a process comprising the steps of(i) extracting mycelia and fruit bodies of a basidiomycetous fungus,including C. versicolor, with an aqueous solvent, (ii) removingsubstances with a molecular weight of less than 5,000 by ultrafiltrationand/or reverse osmosis; (iii) saturating the extract solution withammonium sulfate; (iv) collecting the resultant precipitate, dissolvingthe precipitate in water, and desalting same; (v) passing the desaltedsolution through a column packed with an ion exchanger; (vi)concentrating and drying the solution to obtain the claimedpolysaccharide.

German Patent DE 2,731,570 and related U.S. Pat. No. 4,140,578 describenitrogen-containing polysaccharides having anti-cancer propertiesproduced by a multi-step process involving cultivating a fungus from thegenus Coriolus in an aqueous culture medium by submerged cultivation,drying the culture media together with the mycelia, producing an extractfrom the dried substance with water or an aqueous alkaline solutionfollowed by ultrafiltration or reverse osmosis to remove substanceshaving a molecular weight of less than 5,000.

Japanese Patent JP 52,083,996 and related U.S. Pat. Nos. 4,289,688 and4,271,151 describe oral and intraperitoneal administration ofprotein-bound polysaccharides having an antitumor effect. Theprotein-bound polysaccharides are obtained by a process comprising thesteps of concentrating an extract obtained from the mycelia and/or fruitbodies of a species of fungi belonging to the genus Coriolus in anaqueous solution, performing consecutive ammonium sulfateprecipitations, followed by dialysis to remove salts, and finallyspray-drying the desalted solution to obtain the claimed protein-boundpolysaccharides.

Japanese Patent JP 09,309,842 describes an antitumor glycoproteincontaining β-1,3-glucan in an amount of less than 0.001 mg/g with amolecular weight of 5,000-1,000,000 kDa from an extract of hyphae andfruiting body of Basidiomycota belonging to the genus Coriolus. Theextract is obtaining by oxidation with periodic acid or its salt,followed by addition of a reducing agent, removal of low molecularcompounds, and addition of β-1,3-glucanase.

Japanese Patent JP 49,048,896 describes soluble phosphorylated glucansfrom C. versicolor having a phosphorylated poly-[β-(1-3)glucopyranose]chain and its use in dermal wound healing, specifically as an agentimpregnated into bandage, suture or dressing.

U.S. Pat. No. 7,048,932 describes compositions and methods forstimulating the immune system comprising administering a purifiedextract of C. versicolor having a molecular weight of 0.3 kDa to 5 kDaas determined by size exclusion chromatography and comprising at leastone peptide-linked glucan in which the glucose molecules of the glucanare linked by a (1→3) linkage,. The purified extract taught in the '932Patent is prepared by the steps of (i) treating C. versicolor withalkali; (ii) separating a supernatant; (iii) subjecting the supernatantto (a) cationic exchange followed by (b) anionic exchange; and (iv)collecting a fraction comprising the peptide-linked glucan.

A series of related patents assigned to Active Organics, LP—U.S. Pat.Nos. 5,976,556; 6,569,437; and 6,656,701—describe the uses of one ormore acid protease enzymes in combination with an acidic bufferingsystem that enhances epidermal exfoliation and/or epidermal cellrenewal, thereby improving the texture or appearance of the skin.

Extracts of Rhizomucor miehei are commercially-available from a numberof sources, including Novozymes, Inc. (Franklinton, N.C.), ValleyResearch (South Bend, Ind.) and Active Organics LP (Lewisville, Tex.),exhibit enzymatic activity, principally from acid proteases.

US Patent Application Publication No. 2007/0160563 discloses topicalcompositions comprising extracts of R. miehei that are substantiallydevoid of acid-protease activity and their use in treating dermatologicconditions, including reducing the appearance of signs of skin aging.

SUMMARY OF THE INVENTION

The present invention relates to an acid protease hydrolyzedpolysaccharopeptide extract of C. versicolor that is substantiallydevoid of acid protease activity having anti-inflammatory properties asmeasured in vitro (by reduction in the level of expression of the PTGS2gene as analyzed using DNA microarrays) and in vivo.

DETAILED DESCRIPTION OF THE INVENTION

Acid protease hydrolyzed polysaccharopeptide extract of C. versicolorthat is substantially devoid of acid protease activity iscommercially-available from the Active Organics LP under the tradenameActisoothe™ and is produced as follows: An initial fraction of C.versicolor is produced by mixing the macerated fruiting body of themushroom in ethanol at a ratio of from about 1:5 to about 1:3,preferably about 1:4, for a period of from about 36 hours to about 60hours, preferably for a period of about 48 hours. The resultingprecipitate is collected and mixed with an acidic aqueous solvent,having a pH of from about 3.5 to 4.5, preferably from about 3.8 to about4.2. The acidic aqueous solvent is made by mixing (i) citric acid (3%)and (ii) glycerin or a lower glycol, preferably propylene glycol orbutylene glycol (10%) with (iii) deionized water and adjusting the pH tothe desired range with a 25% solution of sodium hydroxide.

Preferably, the precipitate is present in the acidic aqueous solvent ina ratio of from about 1:50 to about 1:9, more preferably from about 1:40to about 1:20. Deionized water is added QS. This precipitate is furtherextracted for period of from about 36 hours to about 60 hours,preferably for a period of about 48 hours, at a temperature of fromabout 30° C. to 50° C., more from about 35° C. to about 45° C.

Following this extraction period, the water-soluble polysaccharopeptideproteinaceous fraction is hydrolyzed by addition of an acid protease,preferably an acid protease of R. miehei, still more preferably an acidprotease of R. miehei having from 4,000 to about 10,000 HUT units ofactivity per milliliter. A preferred acid protease of R. miehei isavailable under the tradename Actizyme® 3M-M from Active Organics LP.The acid protease is added as solution of from about 0.001% to 10%,preferably from about 0.01% to about 3% and more preferably from about0.5% to about 1.5%. Hydrolysis is then conducted at a temperature offrom about 35° C. to about 45° C., preferably at about 40° C., for aperiod of from about 36 hours to about 60 hours, preferably for a periodof about 48 hours. Following hydrolysis, the acid protease is removed orinactivated by techniques well-known to those having ordinary skill inthe art, including by molecular weight sieve, thermal inactivationand/or pepstatin-affinity gel chromatography.

According to one aspect of the present invention, the acid proteasehydrolyzed polysaccharopeptide extract of C. versicolor that issubstantially devoid of acid protease activity is present in adermatologically-acceptable carrier at a concentration of from about0.01% to about 50%, preferably at a concentration of from about 0.1% toabout 10%, still more preferably at a concentration of from about 0.25%to about 5%.

One preferred aspect of the present invention is directed to topicalphotoprotective and anti-aging products comprising the acid proteasehydrolyzed polysaccharopeptide extract of C. versicolor that issubstantially devoid of acid protease activity at a concentration of atleast about 0.1%. By photoprotective products are meant topicalformulations that provide protection from ultraviolet radiation. Byanti-aging products are meant topical formulations that help reduce theappearance of fine lines, wrinkles, pigment discoloration associatedwith chronological or environmental aging.

In Vitro Anti-Inflammatory Activity—DNA Microarray Analysis

The anti-inflammatory effect of acid protease hydrolyzedpolysaccharopeptide extract of C. versicolor that is substantiallydevoid of acid protease activity is measured based on changes in thelevel of expression of the PTGS2 gene analyzed using DNA microarrays asdescribed below.

Epidermal full-thickness tissue, supplied by MATEK Corporation,(Ashland, Mass.) is used for in vitro testing. Tissue samples areremoved from the shipping tray, placed into a 6-well plate containing2.5-5.0 ml of assay medium (37±2° C.), and incubated for at least 24hours at 37±2° C. and 5±1% CO2. After this initial incubation, the assaymedium is replaced with 2.5-5.0 ml of fresh medium (37±2° C.). 25-50 mlof test material (test sample) and/or phosphate buffered saline(negative control) is then applied directly onto the surface of thetissue. The 6-well plates are then incubated at 37±2° C. and 5±1% CO2for 24 hours. Thereafter, the tissue samples are washed at with 100 mlof PBS and placed into a 1.5 ml centrifuge tube containing 10-12 volumesof guanidinium thiocyanate lysis solution. The tissues are minced withfine tipped scissors and homogenized until thoroughly disrupted. Afterhomogenization, the tissues are centrifuged at 15,000 RPM for 10minutes. The supernatant is transferred to a new tube. The pellet(tissue debris) is discarded and the tissue homogenate is then stored at−75° C. until the RNA extraction process (described below) is completed.

RNA Isolation

RNA isolation was performed using the RNAqueous Kit from Ambion Inc.(Austin, Tex.). To the cell lysates or tissue homogenates preparedabove, an equal volume of 64% ethanol is added and the tubes arevortexed. Up to 700 ml of the resulting mixture is transferred to aglass fiber filter cartridge, which is loaded into a 1.5 ml collectiontube and the cartridge is centrifuged for 1 minute at 14,000 RPM. Theflow-through is discarded. The remaining mixture is loaded into thefilter cartridge and the centrifugation process is repeated until all ofthe mixture is processed. The filter is then washed to remove anyresidual cellular debris from the RNA bound to the glass fibers byapplying 700 ml of a first wash solution (1 time) and 500 ml of a secondwash solution (2 times) to the filter cartridge and centrifuging at14,000 RPM for 1 minute to pass each wash through the cartridge. Theflow-through is discarded after each wash. After the final wash, onefinal spin is performed without wash solution to remove any residualwash solution in the filter cartridge. The RNA bound to the glass fiberswithin the cartridge is then eluted by applying 30 ml of Tris-EDTAbuffer (Sigma) (10 mM Tris-HCl, 1 mM EDTA (Sigma), preheated to 70-80°C., hereinbelow “TE buffer”) to the cartridge and centrifuging thecartridge in a new collection tube at 14,000 RPM for one minute. Forsamples prepared from cell lysates and small tissues, the elutionprocess is repeated with an additional 30 ml of preheated TE buffer. Forsamples prepared from larger (i.e., full thickness) tissues, the elutionprocess is repeated two additional times. After the RNA is eluted, RNAconcentration is quantified using a Ribogreen assay. RNA quality isassessed via gel electrophoresis.

RNA Concentration Assay

Ribogreen reagent (NanoDrop Technologies, Wilmington, Del.) is providedas a stock solution in DMSO. Prior to use, the reagent is diluted 2000fold in TE buffer. The RNA assay requires 200 ml of diluted Ribogreenreagent per sample to be tested and 1 ml of reagent as a standard. Onceprepared, the diluted reagent is stored protected from light. A seriesof RNA standards are prepared by diluting purified ribosomal RNA derivedfrom E. coli to the following concentrations: 2 mg/ml, 1 mg/ml, 200ng/ml, 40 ng/ml and 0 ng/ml (blank). Prior to assaying, the RNA samplesprepared above are diluted 1000 fold in TE buffer. For the RNA assay,100 ml of the diluted samples or standards are transferred to the wellsof a black 96-well plate. The samples and standards are assayed induplicate. After the samples/standards are added to the plate 100 ml ofdiluted Ribogreen assay reagent is added to the wells and the plate isgently mixed and allowed to incubate for 5-10 minutes protected from thelight. After this incubation, the plate is read with a fluorometer (ColeParmer) using an excitation wavelength of 500 nm and an emissionwavelength of 525 nm.

RNA Gel Electrophoresis

A 1% RNA gel is prepared by adding 0.3 g agarose to 21.6 mldiethylpyrocarbonate (DEPC) treated water. The agarose is dissolved byboiling the water in a microwave oven. After the solution is cooled toapproximately 55° C., 5.4 ml of formaldehyde and 3.0 ml 10×MOPS(3-morpholinopropanesulfonic acid) (0.2 M MOPS [pH 7.0], 20 mM sodiumacetate, 10 mM EDTA, made in DEPC H₂O) is added and filter sterilized.After mixing, the agarose gel is cast in the horizontal gel apparatuswith loading slots placed on the side of the gel closest to the negativeterminal. The gel is allowed to set for at least 1 hour at roomtemperature. While the gel is setting, 175 ml of 1×MOPS is prepared bydiluting the 10× stock. After the gel is set, the comb is removed andthe buffer chamber of the gel apparatus is filled with 150-175 ml 1×MOPS(enough buffer is added to cover the gel with approximately 3 mm ofbuffer). The cover is placed on the apparatus, the electrical leads areattached to the power source, and the empty gel is run at 40 V (4 V/cm)for 5-10 minutes. While the gel is running, the RNA samples are preparedby transferring approximately 1 mg of each sample RNA to a 600 ml PCRtube. DEPC H₂O is used to bring the total volume of all the samples to acommon level and then 1-3 volumes of a gel-loading buffer (i.e. 5%glycerol, 1 mM EDTA, 0.025% bromophenol blue, 0.025% xylene cyanol FF,20% formaldehyde, 50% formamide, 10 mg/ml ethidium bromide) are added.The samples are denatured by placing them at 65-70° C. for 5-15 minutesand then placed on ice to cool. The samples are then carefully loadedinto the lanes (each loading slot can hold 10-15 ml of sample, dependingupon the thickness of the gel) and run on the gel at 40 V for 1-3 hours.At the end of the run, the RNA is visualized by placing the gel on a UVtransilluminator (Cleaver Scientific). An RNA sample is used forsubsequent processing if both the 18S and 28S ribosomal bands areclearly visible and there is little or no staining below the 18S band.

mRNA Amplification

mRNA is amplified using the MessageAmp, aRNA kit from Ambion Inc.(Austin, Tex.) as follows:

First Strand cDNA Synthesis

To start the first strand synthesis, 5 mg of total RNA for each sampleare added to 600 ml PCR tubes and the total volume of liquid in the tubeis adjusted to 12 ml with DEPC H2O. To each tube, 1 ml of T7 Oligo(dT)primer is added and the tube is incubated at 70±2° C. for 10 minutes todenature the RNA and is then placed on ice to allow the primer to annealto the poly A ends of the mRNA. After cooling, 2 ml of 10×first strandbuffer, 1 ml of RNAse inhibitor and 4 ml of dNTP mix is added to eachtube, and the tube is placed at 42° C. As soon as the tube is heated, 1ml of reverse transcriptase is added and the tubes are returned to 42±2°C. for 2 hours. At the end of the two hours, the tubes are brieflycentrifuged to collect all of the fluid at the bottom of the tube andthen placed on ice.

Second Strand Synthesis and cDNA Purification

For the synthesis of the second strand of cDNA the following ingredientsare added sequentially to the tubes: 63 ml DEPC H₂O, 10 ml 10× secondstrand buffer, 4 ml dNTP mix, 2 ml DNA Polymerase and 1 ml of RNAse H.The tube is mixed and then incubated at 16±2° C. for 2 hours. Towardsthe end of the 2 hour incubation, a sufficient quantity of DEPC H₂O iswarmed to 50±2° C., and a cDNA purification filter cartridge isequilibrated with 50 ml of cDNA binding buffer (one cartridge persample) for at least 5 minutes. After the samples are finishedincubating, 250 ml of cDNA binding buffer are added to each tube andthoroughly mixed. The contents of the PCR tube are then transferred tothe cDNA purification filter cartridge. The cartridge is then placed ina collection tube and centrifuged at 10,000 RPM for 1 minute. Theflow-through is discarded and 650 ml of cDNA wash solution is added tothe cartridge. The cartridge is centrifuged again, the flow-through isdiscarded, and is then centrifuged one additional time to ensure thatthe wash buffer has been completely emptied from the filter. The cDNA iseluted by applying 10 ml of preheated DEPC H2O to the filter andcentrifuging the filter in a new collection tube at 10,000 RPM for oneminute. This elution is performed one additional time to give a totalvolume of 16-18 ml of cDNA solution.

In Vitro Transcription to Synthesize aRNA and aRNA Purification

In vitro transcription begins by adding the following to the cDNAsolution: 4 ml each of T7 ATP solution, T7 CTP solution, T7 GTPsolution, T7 UTP solution, 4 ml of 10×Reaction buffer, and 4 ml of T7enzyme mix. The tube is mixed and then incubated at 37±2° C. for 6-14hours. Towards the end of the incubation, a sufficient volume of ElutionSolution is warmed to 50-60° C. and an aRNA filter cartridge isequilibrated with 100 ml of aRNA binding buffer for at least 5 minutes.At the end of the incubation period, 350 ml of aRNA binding buffer isadded to the sample tubes and thoroughly mixed. An additional 250 ml ofabsolute ethanol is also added to each tube. The mixture is thentransferred to an aRNA filter cartridge; the cartridge is then insertedinto a collection tube and centrifuged at 10,000 RPM for 1 minute. Theflow-through is discarded and 650 ml of aRNA wash buffer is added to thecartridge followed by centrifuging at 10,000 RPM for one minute. Afterdiscarding the flow-through, the cartridge is spun one final time toremove all traces of the wash buffer. The cartridge is then transferredto a new collection tube. 25 ml of pre-warmed Elution Solution is addedto the cartridge. The cartridge is incubated for 2 minutes at roomtemperature and then aRNA is eluted by centrifuging for 1 minute at10,000 RPM. This elution is performed one additional time to give atotal volume of 45-50 ml of aRNA solution. The final concentration ofthe aRNA is determined by the Ribogreen assay described above. Inaddition, the quality of the aRNA is checked via gel electrophoresis asdescribed above. An aRNA sample is used for subsequent processing if abroad band of RNA is observed.

Labeling and Purification of aRNA

aRNA is labeled with fluorescent dyes using the PerkinElmer ASAP RNALabeling Kit. Two tubes are prepared for the labeling process—for theuntreated sample Cy3 labeling (green), and for the treated sample Cy5labeling (red). To the Cy3 tube add 2 mg of aRNA prepared from theuntreated/control sample and add enough DEPC H2O to bring the totalvolume up to 4 ml. To the Cy5 tube add 2 mg of aRNA prepared from thesample treated with the test material and add enough DEPC H2O to bringthe total volume up to 4 ml. To both tubes, add 5 ml of ASAP labelingbuffer and 1 ml of the specific dye for the tube (Cy3 or Cy5). Incubatethe tubes for 15 minutes at 85±2° C. At the end of the 15 minutes, placethe tubes on ice to cool and then add 2.5 ml of ASAP stop solution toeach tube. The above proportions are sufficient for analyzing onemicroarray chip. If more chips are to be used then the labeling isincreased proportionately.

To purify the labeled aRNA, a microcon YM-30 filter column is insertedinto a collection tube and filled with 400 ml of TE buffer. The Cy3 andCy5 probes are combined (12.5 ml of each) and then added to the microconfilter and thoroughly mixed with the TE buffer. The filter iscentrifuged at 12,000 RPM for 8 minutes and the flow-through isdiscarded. The column is washed twice with 400 ml of TE buffer,discarding the flow though each time. After the final wash, the filtercolumn is inverted, placed into a new collection tube and centrifuged at12,000 RPM for 2 minutes to collect the probe (the probe is concentratedin a volume of 2-30 ml of residual TE buffer).

Microarray Hybridization and Washing

For hybridization, 45 ml of 10× control target RNA (supplied withAgilent Technologies In Situ Hybridization Kit) is mixed with 160 ml ofDEPC H₂O and 9 ml of 25× Agilent Fragmentation Buffer. This mixture isincubated at 60° C. for approximately 30 minutes in a hybridizationoven. At the end of the incubation, 225 ml of Agilent HybridizationBuffer is added along with the fluorescent aRNA probes prepared above.The mixture is then incubated at 70° C. for 5-10 minutes in a waterbath.During this incubation period, an Agilent SUREHYB hybridization chamberis prepared by inserting a glass gasket slide into the bottom half ofthe chamber. At then end of the incubation, the hybridization mixture(approximately 450 ml) is applied to the glass gasket slide and anAgilent Human 1A Oligo Microarray Chip is placed face down on top of thegasket such that the hybridization solution is sandwiched between theglass gasket slide and the microarray face of the chip. The top half ofthe chamber is attached and the connecting thumbscrew tightened. Afterverifying that there is good bubble formation in the chamber, it isplaced into the hybridization oven for approximately 17 hours (60° C.and rotating at 4 RPM). At then end of the hybridization period, themicroarray/glass gasket is removed from the SUREHYB chamber and placedin 50 ml of a first wash solution (room temperature, 6×SSC, 0.005%Triton X-102). After the gasket has fallen away from the microarray, thearray is transferred to 300 ml of fresh wash solution 1 on a magneticstir plate. The array is washed while the solution is mixed at mediumspeed for 10 minutes and is then transferred to 300 ml of wash solution2 (0.1×SSX, 0.005% Triton X-102, 4° C.) for 5 minutes. After the finalwash, the array is centrifuged at 500 RPM for 5 minutes until dry.

Microarray Scanning and Analysis

The microarrays are scanned with an Axon GenePix 4100A Scanner with thescanning resolution set to 10 mm and analyzed with GenePix Pro software.During the initial scan the PMT gains for the scanner are adjusted suchthat the Cy5 /Cy3 image count ratios are between 0.88 and 1.12.

To derive the standard curve for the Ribogreen assay, the relativefluorescent units (RFU) versus the known RNA concentrations in mg/ml forthe standards is plotted and subjected to regression analysis toestablish the line that best fits these data points. Mean RFU values forthe test materials and untreated samples are then used to estimate theamount of RNA present in each sample. The level of gene expression isrelated to the fluorescence intensity of the probed gene marker on themicroarray. Fluorescence measurements between the the Cy3 and Cy5 probesare normalized. The total fluorescent signal for both dyes is normalizedwith a correction factor such that the ratio of total intensities forboth dyes equal to one.

Criteria for evaluating changes in gene expression are known to those ofordinary skill in the art and include the following: (i) the ratio ofCy3 /Cy5 (untreated/treated) fluorescence intensity is greater than 1.5or less than 0.66, corresponding to a change in gene expression of atleast +/−30%; (ii) the fluorescence intensity of the gene marker isgreater than the background intensity; (iii) the gene feature is clearlymarked specifically by the aRNA probes and is not due to non-specificfluorescence. The first two criteria are filtered via computer analysis.The last criterion requires visual inspection of the array.

Cy3 /Cy5 ratios of greater than about 1.3 are interpreted to indicatethat a gene is upregulated by the treatment, whereas ratios of less thanabout 0.7 are interpreted to indicate a downregulated gene. Thus, aratio of 1.3, where the treated value is 130% of the untreated value,indicates a 30% increase in gene expression. Similarly, a ratio of 0.7means that the treated value was 70% of the untreated value, indicatinga 30% decrease in gene expression.

EXAMPLE 1

Two polysaccharopeptide extracts of C. versicolor—the first, ahydrolyzed with acid protease of R. miehei and thereafter renderedsubstantially devoid of acid protease activity according to the methodsdescribed above, the second a non-hydrolyzed extract—were tested for theability to reduce the level of expression of PTGS2. The acid proteasehydrolyzed extract reduced the expression of mRNA coding for PTGS2 byabout 67%.

In Vivo Anti-Inflammatory Efficacy

EXAMPLE 2 Reduced Erythema After UV Exposure

A gel comprising 2% by weight of extract of C. versicolor according tothe present invention (hydrolyzed with acid protease of R. miehei andthereafter rendered substantially devoid of acid protease activity) andthickened with Carbopol 940, having a pH of about 6.5 was appliedtopically (either five minutes before or after UVR exposure) to 20 humansubjects at a dose of 2-4 mg/cm². In the examples that follow, theextract of C. versicolor according to the present invention is referredto by its tradename, Actisoothe™. The subjects were then exposed sixtimes over an eight-day period to 1 MED of ultraviolet radiation (“UVR”)according the methodology set out in the by the FDA in the finalmonograph for sunscreen drug products for over-the-counter human use aspublished in Federal Register Vol. 64, No. 98, pp. 27666-27693 (May 21,1999). One MED is the quantity of erythema-effective energy required toproduce the first perceptible, redness reaction with clearly definedborders, Reduction in erythema was observed based on a* value readingsfrom a Minolta Chroma Meter. The data presented are increases in a*values compared to pre-UVR exposure values.

Erythema Erythema % Reduction (day 4) (day 8) (day 8) Control 7.9 12.4NA 2% Actisoothe ™ before UV 3.4 6.5 52% 2% Actisoothe ™ after UV 5.28.2 34%

EXAMPLE 3 Reduction of Stinging Response (Lactic Acid)

On twenty subjects 8% lactic acid in 80:20 ethanol:water mixture (pH 3)was applied in excess to the nasal fold area with a Q-tip typeapplicator. Stinging responses of the control group (no C. versicolorapplied prior to acid application) were compared to that of the testgroup (2% C. versicolor applied). Stinging was evaluated subjectively ona 0-4 scale over a 20 minute time period (time=0, 1, 2, 5, 8, 14, 20minutes). Scores were summed and presented as initial and delayed sting:

Test Panel Treatment Initial Delayed Total Sting % Reduction Controlnone 2.3 4.9 7.2 NA Control 2% Actisoothe 2.2 4.2 6.4 11%

EXAMPLE 4 Reduction of Erythema (Balsam of Peru)

Balsam of Peru, a know contact irritant, was applied to the skin ofsubjects. Erythema was measured using a Minolta Chroma Meter (a* value).After five days of treatment with the 2% (Actisoothe™), the test group(C. versicolor applied) showed an increased resistance to Balsam of Peruapplication of 25% over the control group (no C. versicolor applied).

Test Panel Treatment Increase in Erythema (Δ*a) % Reduction Control none44.5 NA Test 2% Actisoothe 32.3 25%

EXAMPLE 5 Reduction of Skin Reactivity (Methyl Nicotinate)

Skin reactivity, measured as an erythemic response (measured as visuallyidentifiable redness) was tested by topical application of methylnicotinate (Sigma Aldrich) in increasing concentrations until ameasurable erythemic response was observed. Test groups (2% C.versicolor applied) required a 25% increase in dose of methyl nicotinateto induce an erythemic response than did the control group (no C.versicolor applied).

Concentration Inducing Test Panel Treatment Erythema % Change Controlnone 0.084 NA CIS Group none 0.025 NA Control 2% Actisoothe ™ 0.103 +25% CIS Group 2% Actisoothe ™ 0.065 +160%

EXAMPLE 6 Reduction of Cosmetic Intolerance Syndrome (CIS)

Test subjects with CIS, self identified and exhibiting at least onemoderate to severe cosmetic product reaction (cosmetic acne, productrelated erythema, transitory itching or burning, allergic response, orcontact dermatitis) within the past six months were recruited for thisstudy. While the CIS group did not have a significantly higher rate oftransepidermal water loss (TEWL) rate compared to the control theyexhibited a more pronounced response when exposed to a topicalirritant—specifically 8% lactic acid pH 3 applied in excess to the nasalfold area in an 80:20 ethanol:water vehicle. Stinging was evaluatedsubjectively on a 0-4 scale every minute for 5 minutes, thereafter everythree minutes until 20 minutes (e.g., at time=8, 11, 14, 17 and 20minutes). Scores were summed and presented as initial and delayed stingscores. After five days of treatment with a 2% Actisoothe™ gel (at adose of 5 mg product/cm² twice a day) the CIS group showed a 31%reduction in sting response:

Test Panel Treatment Initial Delayed Total Sting % Reduction Controlnone 2.3 4.9 7.2 NA CIS Group none 4.3 9.1 13.4 NA Control 2% Actisoothe2.2 4.2 6.4 11% CIS Group 2% Actisoothe 3.3 6.1 9.4 31%

Anti-inflammatory compositions according to the present invention mayoptionally include (in addition to extract of C. versicolor hydrolyzedwith acid protease of R. miehei and thereafter rendered substantiallydevoid of acid protease activity) one or more steroidal or non-steroidalanti-inflammatory drug (NSAID) known to those of ordinary skill in theart. Preferred examples of NSAIDS include propionic acid derivatives,acetic acid derivatives, fenamic acid derivatives, biphenylcarboxylicacid derivatives, and oxicams. The Cosmetic, Toiletries & FragranceAssociation, International Cosmetic Ingredient Dictionary and Handbook,Vol. 11, p. 1364 (11^(th) Edition, 2006) (“CTFA Dictionary”) describes awide variety of non-limiting cosmetic and pharmaceutical ingredientscommonly used in the skin care industry, which are also suitable for usein combination with the extract of C. versicolor hydrolyzed with acidprotease of R. miehei and thereafter rendered substantially devoid ofacid protease activity as claimed in the present application.

The following formulation examples are further illustrative of thepresent invention. The components and specific ingredients are presentedas being typical, and various modifications can be derived in view ofthe foregoing disclosure within the scope of the invention. Allpercentages, ratios and proportions herein are by weight, unlessotherwise specified. All temperatures are in degrees Celsius unlessotherwise specified.

FORMULATION EXAMPLE 1 Toner

Deionized Water 93.190% Methyl Gluceth-20 1.000% Potassium Sorbate0.100% Sodium Benzoate 0.100% Phenoxyethanol 0.600% Citric acid 0.010%Extract of C. versicolor hydrolyzed with acid protease of 5.000% R.miehei and thereafter rendered substantially devoid of acid proteaseactivity

Add ingredients sequentially in order listed. Mix until clear. Endprocessing.

FORMULATION EXAMPLE 2 Face Cream

Part A Deionized Water 62.600% Magnesium Aluminum Silicate 0.400%Xanthan Gum 0.150% Acrylates/C₁₀₋₃₀ Alkyl Acrylate Crosspolymer 0.750%Part B Butylene Glycol 4.000% Disodium EDTA 0.050% Part C HydrogenatedLecithin 0.500% Caprylic/Capric Triglyceride 8.000% Simmondsia Chinensis(Jojoba) Seed Oil 5.000% Octyl Palmitate 4.000% Cetearyl Alcohol 2.000%PEG-8 Stearate 1.000% PEG-100 Stearate 0.800% Part D Triethanolamine 99%0.100% Part E Aloe Barbadensis Leaf Juice (Activera ® 10x, 5.000% ActiveOrganics) Phenoxyethanol 0.500% Potassium Sorbate 0.100%Methylisothiazolinone 0.050% Extract of C. versicolor hydrolyzed withacid protease of 5.000% R. miehei and thereafter rendered substantiallydevoid of acid protease activitySprinkle Magnesium Aluminum Silicate, Xanthan Gum, Acrylates/C₁₀₋₃₀Alkyl Acrylate Crosspolymer into vortex of water. Mix and heat to 80° C.Add Part B to Part A, mix and hold the temperature at 80° C. In aseparate vessel, mix Part C and heat to 75° C., mix until clear. AddPart C to Parts A and B, mix for 10 minutes. Add Part D to Parts ABC.Mix for 15 minutes. Switch to sweep mixing. Cool batch to 45° C. In aseparate container, add ingredients in Part E. Mix until uniform. At 45°C., add Part E, to Parts ABCD. Mix and cool to 25° C. End processing.

FORMULATION EXAMPLE 3 Eye Cream

Part A Deionized Water 57.650% Acrylates/C10-30 Alkyl AcrylateCrosspolymer 0.300% Panthenol 0.100% Potassium Sorbate 0.100% DisodiumEDTA 0.100% Allantoin 0.100% Part B Caprylic/Capric Triglyceride 2.000%Dimethicone 3.000% Butyrospermum Parkii (Shea Butter) 2.000% CarthamusTinctorius (Safflower) Seed Oil 2.000% Cetearyl Alcohol 1.500%Dimethiconol 1.300% Steareth-2 1.000% Steareth-21 0.500% Cyclomethicone5.000% Part C Triethanolamine 0.250% Part D Carbomer 940 2% Solution10.000% Part E Mucor Miehei Extract, Butylene Glycol, and 5.000%N-Acetylglucosamine Part F Sodium Hyaluronate (Actimoist ® Bio 2, ActiveOrganics) 2.000% Phenonip 1.000% Aloe Barbadensis Leaf Juice (Activera ®10X, 5.000% Active Organics) Tocopherol 0.100%Sprinkle Acrylates/C₁₀₋₃₀ Alkyl Acrylate Crosspolymer into vortex ofwater. Mix and heat to 75° C. Mix and heat Part B to 70° C. Add Part Bto Part A, mix for 10 minutes. Add Part C. Mix for 10 minutes. Add PartD. Mix and cool to 45° C. At 45° C., add Parts E and F. Mix and cool to25° C. End processing.

FORMULATION EXAMPLE 4 Lipstick

Part A Ricinus Communis (Castor) Seed Oil 24.37% Octyl Palmitate 33.33%Petrolatum 10.84% Beeswax 3.33% Paraffin Wax 3.33% Euphorbia Cerifera(Candelilla) Wax 5.20% Ozokerite 3.00% Copernicia Cerifera (Carnauba)Wax 2.50% Simmondsia Chinensis (Jojoba) Seed Oil 8.00% Propylparaben0.10% Part B Polyglyceryl-4 Isostearate 1.00% Mucor Miehei Extract,Butylene Glycol, and 5.00% N-AcetylglucosamineMix and heat Part A to 80° C. Pre-mix Part B; add to Part A. Mix andpour into container.

FORMULATION EXAMPLE 5 Face Mask

Part A Deionized Water 59.960% Aloe Barbadensis Leaf Juice (Activera ™10X, 5.000% Active Organics) Glycerin 4.000% Caffeine 0.100% Acacia Gum0.300% Chromium Oxide Green 0.500% Titanium Dioxide 3.000% Methylparaben0.200% Part B Glyceryl Stearate 6.000% Simmondsia Chinensis (Jojoba)Seed Oil 1.500% Tocopheryl Acetate 0.100% Propylparaben 0.100% Part CBentonite 11.000% Part D Phenoxyethanol 0.500% Citric Acid 50% 2.100%Extract of C. versicolor hydrolyzed with acid protease of 5.000% R.miehei and thereafter rendered substantially devoid of acid proteaseactivity Part E Essential Oil (Spearmint) 0.070% Essential Oil(Peppermint) 0.070%Mix and heat Part A to 75° C. Mix and heat Part B to 75° C. HomogenizePart A, then add Part B continuing, mixing in the homogenizer for 5minutes. Start to cool. At 60° C., add Part C; mix well. Continuecooling. At 45° C., add Parts D and E. Mix and cool to 25° C. Endprocessing.

FORMULATION EXAMPLE 6 Moisturizing Shampoo

Part A Deionized Water 46.680% Aloe Barbadensis Leaf Juice (Activera ®10X, 10.000% Active Organics) Part B Sodium C14-16 Olefin Sulfonate18.000% Cocamidopropyl Betaine 18.000% Glucamate DOE-120 1.000% Part CPhenoxyethanol 0.300% Kathon CG 0.020% Sodium Chloride 25% Solution qsButylene Glycol and Spiraea Ulmaria Extract (Actiphyte ® 1.000% Queen ofMeadow Concentrate, Active Organics) Part D Extract of C. versicolorhydrolyzed with acid protease of 5.000% R. miehei and thereafterrendered substantially devoid of acid protease activityMix and heat Part A to 50° C. Add Part B to Part A; mix until clear. AddParts C and D to Parts A and B. Mix and cool to 25° C. End processing.

FORMULATION EXAMPLE 7 Moisturizing Conditioner

Part A Deionized Water 64.670% Aloe Barbadensis Leaf Juice (Activera ®10X, 5.000% Active Organics) Panthenol 0.200% Part B Jojoba Oil 2.000%Behentrimonium Methosulfate, Cetearyl Alcohol 4.000% StearamidopropylDimethylamine 2.000% Cetearyl Alcohol 4.500% PEG-100 Stearate 0.880%Glyceryl Stearate 1.200% Part C Water, Phenyl Trimethicone,Cyclomethicone, 10.000% Polysilicone-11, Lecithin (Actiprime ™ 100,Active Organics) Part D Extract of C. versicolor hydrolyzed with acidprotease of 5.000% R. miehei and thereafter rendered substantiallydevoid of acid protease activity Phenoxyethanol 0.500%Methylisothiazolinone 0.050%Mix and heat Part A to 75° C. Mix and heat Part B to 75° C. Add Part Bto Part A. After mixing, add Part C and mix. Cool until 45° C., then addPart D. Mix and cool to 25° C. End processing.

FORMULATION EXAMPLE 8 Face Serum

Part A Deionized Water 80.850%  Keltrol RD 0.250% Butylene Glycol 0.400%Part B Water 0.600% Potassium Sorbate 0.100% Part C Water, AlgaeExtract, and Aloe Barbadensis Leaf Juice  5.0% (Actisea ® 100, ActiveOrganics) Part D Aloe Barbadensis Leaf Juice (Activera ® 10x, 5.000%Active Organics) Phenoxyethanol 0.600% Neolone 950 0.050% Extract of C.versicolor hydrolyzed with acid protease of 5.000% R. miehei andthereafter rendered substantially devoid of acid protease activity PartE Water 2.000% Allantoin  0.1% Disodium EDTA  0.05%Mix Part A. Add pre-dissolved Part B; mix until uniform. Add Part C; mixuntil uniform. Add Part D; mix well. Add pre-dissolved Part E, mix untiluniform. End processing.

While the illustrative embodiments of the invention have been describedwith particularity, it will be understood that various othermodifications will be apparent to and can be readily made by thoseskilled in the art without departing from the spirit and scope of theinvention. Accordingly, it is not intended that the scope of the claimsappended hereto be limited to the examples and descriptions set forthhereinabove but rather that the claims be construed as encompassing allthe features of patentable novelty which reside in the presentinvention, including all features which would be treated as equivalentsthereof by those skilled in the art to which the invention pertains.

1. A topical anti-inflammatory mushroom hydrolysate substantially devoidof acid protease activity produced by the steps of (a) extractingCoriolus versicolor in an acidic aqueous solvent having a pH of fromabout 3.5 to about 4.5 to form a water-soluble polysaccharopeptideextract, (b) hydrolyzing the water-soluble polysaccharopeptide extractwith an acid protease enzyme of Rhizomucor miehei and, (c) inactivatingor removing the acid protease via molecular weight sieve, thermalinactivation and/or pepstatin-affinity gel chromatography.
 2. Thetopical anti-inflammatory mushroom hydrolysate substantially devoid ofacid protease activity according to claim 1 wherein the acid proteaseenzyme of Rhizomucor miehei used to hydrolyze the water-solublepolysaccharo-peptide extract of Coriolus versicolor has from about 4,000to about 10,000 HUT units of activity per milliliter.
 3. Adermatocosmetic composition comprising from about 0.01% to about 10% byweight of the topical anti-inflammatory mushroom hydrolysate accordingto claim
 1. 4. A dermatocosmetic composition comprising the topicalanti-inflammatory mushroom hydrolysate according to claim 1, furthercomprising at least one additional topical anti-inflammatory agent. 5.The dermatocosmetic composition of claim 4 wherein the at least oneadditional topical anti-inflammatory agent is a non-steroidalanti-inflammatory drug.
 6. The dermatocosmetic composition of claim 4wherein the at least one additional topical anti-inflammatory agent ishydrocortisone.
 7. A method for inhibiting the production of COX-2enzymes comprising applying to the skin of a mammal atherapeutically-effective amount of the topical anti-inflammatorymushroom hydrolysate according to claim
 1. 8. The method of claim 7,wherein the topical anti-inflammatory mushroom hydrolysate of claim 1 isapplied in a concentration sufficient to reduce the expression of mRNAcoding for PTGS2 by at least about 50%.
 9. The method of claim 7,wherein the topical anti-inflammatory mushroom hydrolysate of claim 1 isapplied in a concentration sufficient to reduce the expression of mRNAcoding for PTGS2 by at least about 67%.